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Supplementary Figure 1 Supplemental Figure 1. Nogin siRNA potentiates BMP2-induced Smad-dependent BMP- signaling MDA-MB-231 breast cancer cells. MDA-MB-231/BREluc.

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Presentation on theme: "Supplementary Figure 1 Supplemental Figure 1. Nogin siRNA potentiates BMP2-induced Smad-dependent BMP- signaling MDA-MB-231 breast cancer cells. MDA-MB-231/BREluc."— Presentation transcript:

1 Supplementary Figure 1 Supplemental Figure 1. Nogin siRNA potentiates BMP2-induced Smad-dependent BMP- signaling MDA-MB-231 breast cancer cells. MDA-MB-231/BREluc cells were transfected with Noggin siRNA or NT siRNA (Dharmacon) 24 hours prior addition of BMPs (50 ng/ml) for 30 hours. $$$ P< using two-way ANOVA. Experiment was performed in triplicate.

2 Supplemental Figure 2. Smad3/4-dependent TGFβ-signaling in MDA-231 breast cancer cells. MDA-MB-231/CAGAluc2 cells were characterized by performing a dose-response with different TGFβ isoforms. N = 2 independent experiments. No significant differences were observed between the TGFβ isoforms. ** P<0.01 using two-way ANOVA. Supplementary Figure 2 ** (all)

3 Supplementary Figure 3 Supplemental Figure 3. Dose response of BMP7 on Smad-dependent TGFβ reporter imaging in MDA-MB-231 breast cancer cells. (a) The effects of BMP7 on TGFβ reporter imaging in MDA-MB-231/CAGAluc2 in the presence and absence of TGFβ 2. (b) Data are shown relative to basal (left) and TGFβ-stimulated (right) conditions. *** P<0.001 vs Co, ### P<0.001 vs TGFβ using oneway ANOVA using a LSD-post hoc test. a b ###

4 TGFβ 2 +BMP7 TGFβ 2 alone ### Supplemental Figure 4. Inhibitory effects of BMP7 on Smad-dependent TGFβ-signaling in MDA-MB-231 breast cancer cells are are time-dependent. MDA-MB-231/CAGAluc2 cells were used to study inhibitory effects of BMP7 on TGFβ-signaling. (a) Co- incubation of BMP7 with TGFβ 2. In the graph below, only the conditions TGFβ 2 +BMP7 co-incubation vs TGFβ 2 alone (set to 100%) are shown. (b) The longer cells are pretreated with BMP7 the stronger the inhibitory resonse on smad-dependent TGF β signaling. In the graph below, TGFβ 2 only is set as (set to 100%). (c) Pretreatment and withdrawel of BMP7 before addition of TGFβ 2 is still able to inhibit Smad-dependent TGFβ-signaling. In all experiments, TGFβ 2 [1 ng/ml]; BMP7 [50 ng/ml]; * P<0.05; **P<0.01; *** P<0.001 vs Co; # P<0.05 vs TGFβ 2 ; ## P<0.01 vs TGFβ 2 ; ### P<0.001 vs TGFβ 2 ; $ P<0.05; $$ P<0.01; $$$ P<0.001 using two-way ANOVA (a) or one-way (b+c) ANOVA with a LSD post-hoc test. Experiments were performed in triplicate. Supplementary Figure 4

5 a b ### Supplementary Figure 5 Supplementary Figure 5 The heterodimer BMP2/7 is more potent than the BMP2 or BMP7 homodimers in inhibiting TGFβ-signaling in MDA-MB-231 breast cancer cells. Smad-dependent TGFβ reporter imaging in MDA-MB-231/CAGAluc2 cells after stimulation with either TGFβ 1 (a) or TGFβ 3 (b) and co- incubation with BMP7 for 48 hours. Experiments were performed in triplicate. *** P<0.001 vs Co; ## P<0.01 vs TGFβ; ### P<0.001 vs TGFβ; $ P<0.05 vs BMP2; $$ P<0.01 vs BMP2; $$$ P<0.001 vs BMP2; One-way ANOVA with LSD post-hoc testing.

6 72 Hours 48 0 TGFβ 2 (1ng/ml) BMP7 (50ng/ml) 0 TGFβ 2 + BMP7(0hr) TGFβ 2 BMP7(48hr) Control TGFβ 2 + BMP7(48hr) TGFβ 2 + BMP7(24hr) TGFβ 2 + BMP7(6hr) (hrs of pre-incubation) ### *** ### Supplementary Figure 6 Luc. activity [fold induction] Supplemental Figure 6. Inhibitory effects of BMP7 on Smad-dependent TGFβ-signaling in MDA-MB-231 breast cancer cells are are time-dependent. MDA-MB-231/CAGAluc2 cells were used to study inhibitory effects of BMP7 on TGFβ-signaling. (a) Co- incubation of BMP7 with TGFβ 2. In the graph below, only the conditions TGFβ 2 +BMP7 co-incubation vs TGFβ 2 alone (set to 100%) are shown. (b) The longer cells are pretreated with BMP7 the stronger the inhibitory resonse on smad-dependent TGF β signaling. In the graph below, TGFβ 2 only is set as (set to 100%). (c) Pretreatment and withdrawel of BMP7 before addition of TGFβ 2 is still able to inhibit Smad-dependent TGFβ-signaling. In all experiments, TGFβ 2 [1 ng/ml]; BMP7 [50 ng/ml]; * P< 0.05; ** P< 0.01; *** P vs Co; # P< 0.05 vs TGFβ 2 ; ## P< 0.01 vs TGFβ 2 ; ### P< vs TGFβ 2 ; $ P<0.05; $$ P< 0.01; $$$ P<0.001 using two-way ANOVA (a) or one-way (b+c) ANOVA with a LSD post-hoc test. Experiments were performed in triplicate.

7 72 Hours 48 0 TGFβ 2 Control BMP7 *** TGFβ 2 +BMP7 ### BMP7 *** BMP7 $ *** ### $$$ ### New medium TGFβ 2 +BMP7 Luc. activity [fold induction] TGFβ 2 (1ng/ml) BMP7 (50ng/ml) *** Supplementary Figure 7 Supplemental Figure 7. Inhibitory effects of BMP7 on Smad-dependent TGFβ-signaling in MDA-MB-231 breast cancer cells are are time-dependent. MDA-MB-231/CAGAluc2 cells were used to study inhibitory effects of BMP7 on TGFβ-signaling. (a) Co- incubation of BMP7 with TGFβ 2. In the graph below, only the conditions TGFβ 2 +BMP7 co-incubation vs TGFβ 2 alone (set to 100%) are shown. (b) The longer cells are pretreated with BMP7 the stronger the inhibitory resonse on smad-dependent TGF β signaling. In the graph below, TGFβ 2 only is set as (set to 100%). (c) Pretreatment and withdrawel of BMP7 before addition of TGFβ 2 is still able to inhibit Smad-dependent TGFβ-signaling. In all experiments, TGFβ 2 [1 ng/ml]; BMP7 [50 ng/ml]; * P< 0.05; ** P< 0.01; *** P< vs Co; # P< 0.05 vs TGFβ 2 ; ## P< 0.01 vs TGFβ 2 ; ### P< vs TGFβ 2 ; $ P< 0.05; $$ P< 0.01; $$$ P< using two-way ANOVA (a) or one-way (b+c) ANOVA with a LSD post-hoc test. Experiments were performed in triplicate.

8 Supplementary Figure 8 a b Supplemental Figure 8. Effects of the BMP receptor blocker LDN in MDA-MB-231 breast cancer cells. (a) Incubation of MDA-MB-231/BREluc cells with 50ng/ml BMP7 for 48 hours stimulated Smad-dependent BMP-signaling. Treatment with 500 nM LDN two hours prior addition of BMP7 could completely inhibit this response to basal levels. *** P< vs Co; $ P< 0.05 using oneway ANOVA with a LSD post-hoc test. (b) Incubation of MDA-MB-231/CAGAluc2 cells with 1ng/ml TGFβ for 48 hours significantly stimulated Smad-dependent TGFβ-signaling when compared to control. While co-incubation with BMP7 could inhibit Smad-dependent TGFβ-signaling, treatment with 500 nM LDN two hours prior addition of TGFβ and BMP7 could completely block the inhibitory responses of BMP7 on TGFβ-signaling. * P< 0.05 vs TGFβ; $ P< 0.05 using oneway ANOVA with a LSD post-hoc test. Experiments were performed in triplicate.

9 Suppl. Fig. 9. Overexpression of Smad4. TGFβ reporter imaging in MDA-MB- 231/CAGAluc2 cells that overexpress Smad4 (vs Co) before stimulation with TGFβ 2 and co-incubation with BMP7 for 30 hours. *** P< vs Co; # P< 0.05 vs TGFβ 2 ; ### P< vs TGFβ 2 using twoway ANOVA. Supplementary Figure 9

10 Suppl. Fig.10 Smad7 knockdown. (a) Smad7 mRNA levels in MDA-MB-231/CAGAluc2 clones after lentiviral transduction with shSmad7; normalized to GAPDH. N=2 independent experiments. * P<0.05 vs non-targeted control using one-way ANOVA using LSD post hoc test. (b) Subsequently, these clones were challenged with TGF β 2 and BMP7 for 48 hours. The inhibitory responses of BMP7 on TGF β -induced signaling are shown in the lower part. N = 2 independent exp. *** P<0.001 vs Co; # P<0.05 vs TGFβ 2 ; $ P<0.05 vs TGFβ 2 from non-targeted control), using two-way ANOVA. a b Inhibitory Effects of BMP7 on TGFβ-signaling -49%-56%-60% -61% Supplementary Figure 10

11 ALDH -/low a c d b ALDH hi 2 2 Supplementary Figure 11 Supplementary Figure 11 Identification of breast cancer stem cell markers. (a) MDA-MB-231 cells were incubated with ALDEFLUOR substrate and the specific inhibitor of ALDH, DEAB, to define the ALDH -/low and ALDH hi fraction. At basal levels, the ALDH hi fraction was 21.4%. (b) Representative CD24/CD44 contour plots are shown for the ALDH -/low and ALDH hi fraction. The CD44 hi /CD24 -/low fraction is observed in ALDH hi, but not in ALDH -/low fraction in control conditions. Representative CD24/CD44 contour plots for ALDH hi (c) and ALDH -/low (d) fractions under TGFβ 2 and/or BMP2, BMP7, BMP2/7 (co- )stimulated conditions.

12 a b * ** * * # # Supplementary Figure 12 Supplementary Figure 12 ALDH isoforms mRNA expression levels in MDA-MB-231 breast cancer cells. (a) Basal expression levels of ALDH isoforms as detected with qPCR and shown as arbitrary units. (b) Expression levels of the five ALDH isoforms with highest basal levels (see a). Normalized to GAPDH (d.d. CT method) and shown relative to control. Cells were treated for 24hours with either vehicle, BMP2, BMP7 or BMP2/7 (all 50ng/ml). * P<0.05 vs Co; ** P<0.01 vs Co; # P<0.05 vs BMP7 using two- way ANOVA.


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